Nejvíce citovaný článek - PubMed ID 16269704
Cloning, biochemical properties, and distribution of mycobacterial haloalkane dehalogenases
Haloalkane dehalogenases (EC 3.8.1.5) play an important role in hydrolytic degradation of halogenated compounds, resulting in a halide ion, a proton, and an alcohol. They are used in biocatalysis, bioremediation, and biosensing of environmental pollutants and also for molecular tagging in cell biology. The method of ancestral sequence reconstruction leads to prediction of sequences of ancestral enzymes allowing their experimental characterization. Based on the sequences of modern haloalkane dehalogenases from the subfamily II, the most common ancestor of thoroughly characterized enzymes LinB from Sphingobium japonicum UT26 and DmbA from Mycobacterium bovis 5033/66 was in silico predicted, recombinantly produced and structurally characterized. The ancestral enzyme AncLinB-DmbA was crystallized using the sitting-drop vapor-diffusion method, yielding rod-like crystals that diffracted X-rays to 1.5 Å resolution. Structural comparison of AncLinB-DmbA with their closely related descendants LinB and DmbA revealed some differences in overall structure and tunnel architecture. Newly prepared AncLinB-DmbA has the highest active site cavity volume and the biggest entrance radius on the main tunnel in comparison to descendant enzymes. Ancestral sequence reconstruction is a powerful technique to study molecular evolution and design robust proteins for enzyme technologies.
- Klíčová slova
- ancestral sequence reconstruction, haloalkane dehalogenase, halogenated pollutants, structural analysis,
- MeSH
- hydrolasy chemie metabolismus MeSH
- hydrolýza MeSH
- katalytická doména MeSH
- krystalografie rentgenová metody MeSH
- molekulární evoluce MeSH
- molekulární modely MeSH
- Mycobacterium bovis enzymologie MeSH
- proteinové inženýrství metody MeSH
- sekvenční analýza proteinů metody MeSH
- Sphingomonadaceae enzymologie MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- haloalkane dehalogenase MeSH Prohlížeč
- hydrolasy MeSH
Haloalkane dehalogenases (HLDs) have recently been discovered in a number of bacteria, including symbionts and pathogens of both plants and humans. However, the biological roles of HLDs in these organisms are unclear. The development of efficient HLD inhibitors serving as molecular probes to explore their function would represent an important step toward a better understanding of these interesting enzymes. Here we report the identification of inhibitors for this enzyme family using two different approaches. The first builds on the structures of the enzymes' known substrates and led to the discovery of less potent nonspecific HLD inhibitors. The second approach involved the virtual screening of 150,000 potential inhibitors against the crystal structure of an HLD from the human pathogen Mycobacterium tuberculosis H37Rv. The best inhibitor exhibited high specificity for the target structure, with an inhibition constant of 3 μM and a molecular architecture that clearly differs from those of all known HLD substrates. The new inhibitors will be used to study the natural functions of HLDs in bacteria, to probe their mechanisms, and to achieve their stabilization.
- MeSH
- hydrolasy antagonisté a inhibitory chemie MeSH
- inhibitory enzymů chemie izolace a purifikace metabolismus MeSH
- konformace proteinů MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- Mycobacterium tuberculosis enzymologie MeSH
- simulace molekulární dynamiky MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- haloalkane dehalogenase MeSH Prohlížeč
- hydrolasy MeSH
- inhibitory enzymů MeSH
A haloalkane dehalogenase, DpcA, from Psychrobacter cryohalolentis K5, representing a novel psychrophilic member of the haloalkane dehalogenase family, was identified and biochemically characterized. DpcA exhibited a unique temperature profile with exceptionally high activities at low temperatures. The psychrophilic properties of DpcA make this enzyme promising for various environmental applications.
- MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- fyziologická adaptace * MeSH
- hydrolasy chemie genetika metabolismus MeSH
- kinetika MeSH
- koncentrace vodíkových iontů MeSH
- nízká teplota * MeSH
- Psychrobacter enzymologie genetika růst a vývoj fyziologie MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- bakteriální proteiny MeSH
- haloalkane dehalogenase MeSH Prohlížeč
- hydrolasy MeSH
We report the biochemical characterization of a novel haloalkane dehalogenase, DatA, isolated from the plant pathogen Agrobacterium tumefaciens C58. DatA possesses a peculiar pair of halide-stabilizing residues, Asn-Tyr, which have not been reported to play this role in other known haloalkane dehalogenases. DatA has a number of other unique characteristics, including substrate-dependent and cooperative kinetics, a dimeric structure, and excellent enantioselectivity toward racemic mixtures of chiral brominated alkanes and esters.
- MeSH
- Agrobacterium tumefaciens enzymologie genetika metabolismus MeSH
- alkany metabolismus MeSH
- DNA bakterií chemie genetika MeSH
- estery metabolismus MeSH
- hydrolasy genetika izolace a purifikace metabolismus MeSH
- molekulární sekvence - údaje MeSH
- multimerizace proteinu MeSH
- rostliny mikrobiologie MeSH
- sekvenční analýza DNA MeSH
- stereoizomerie MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- alkany MeSH
- DNA bakterií MeSH
- estery MeSH
- haloalkane dehalogenase MeSH Prohlížeč
- hydrolasy MeSH
This study focuses on two representatives of experimentally uncharacterized haloalkane dehalogenases from the subfamily HLD-III. We report biochemical characterization of the expression products of haloalkane dehalogenase genes drbA from Rhodopirellula baltica SH1 and dmbC from Mycobacterium bovis 5033/66. The DrbA and DmbC enzymes show highly oligomeric structures and very low activities with typical substrates of haloalkane dehalogenases.
- MeSH
- Bacteria enzymologie MeSH
- cirkulární dichroismus MeSH
- DNA bakterií chemie genetika MeSH
- hydrolasy chemie genetika izolace a purifikace metabolismus MeSH
- molekulární sekvence - údaje MeSH
- multimerizace proteinu MeSH
- Mycobacterium bovis enzymologie MeSH
- sekvenční analýza DNA MeSH
- substrátová specifita MeSH
- terciární struktura proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- DNA bakterií MeSH
- haloalkane dehalogenase MeSH Prohlížeč
- hydrolasy MeSH
A novel enzyme, DbeA, belonging to the haloalkane dehalogenase family (EC 3.8.1.5) was isolated from Bradyrhizobium elkani USDA94. This haloalkane dehalogenase is closely related to the DbjA enzyme from B. japonicum USDA110 (71% sequence identity), but has different biochemical properties. DbeA is generally less active and has a higher specificity towards brominated and iodinated compounds than DbjA. In order to understand the altered activity and specificity of DbeA, its mutant variant DbeA1, which carries the unique fragment of DbjA, was also constructed. Both wild-type DbeA and DbeA1 were crystallized using the sitting-drop vapour-diffusion method. The crystals of DbeA belonged to the primitive orthorhombic space group P2(1)2(1)2(1), while the crystals of DbeA1 belonged to the monoclinic space group C2. Diffraction data were collected to 2.2 A resolution for both DbeA and DbeA1 crystals.
